ion mobility spectrometry IR spectroscopy

Transcription

1 Debasmita Gho

2 Introducti on Owing to its accuracy, sensitivity, and speed, mass spectrometry (MS) coupled to fragmentation techniques is the method of choice for determining the primary structure of peptides and proteins. To obtain information on higher order structures, condensed-phase spectroscopic or scattering techniques are usually employed. To investigate higher-order structures of peptides and proteins in isolation, mass spectrometry can be coupled to hydrogen/deuterium exchange (HDX) measurements, nonthermal dissociation techniques that rely on the absorption of UV photons or the capture of electrons, ion mobility spectrometry (IMS), or optical/ir spectroscopy.

3 Backgrou nd

4 Backgrou nd Fig 1. Schematic diagram of the experimental setup. Fig 2. IR spectra of a) ubiquitin and b) cytochrome c for various charge states. Gray trace segments for ubiquitin 10+ and 11+ denote a higher photon density. Three types of bands can be identified which are labeled amide IIa, amide IIb, and amide I. c) Relative intensities of the amide

5 In this paper Can the structures of small to medium-sized proteins be conserved after transfer from the solution phase to the gas phase? A combination of ion-mobility mass spectrometry with infrared spectroscopy was used to investigate the secondary and tertiary structure of proteins carefully transferred from solution to the gas phase. The two proteins investigated are myoglobin and β-lactoglobulin, which are prototypical examples of helical and β-sheet proteins, respectively.

6 Experimental setup In IMS biomolecular ions drift through a buffer gas and are separated according to their size, charge and shape. By employing this technique isomers and conformers be separated. Gas-phase spectroscopy on the other hand, provides direct information about the underlying structure of the ion. Vibrational spectroscopy for example, is highly sensitive to the presence of hydrogen bonds and, therefore, yields information about the underlying secondary structure of the molecule.

7 Fig 1. Condensed-phase structures of a) myoglobin, a 153 amino acid protein with a native secondary structure that is mostly α-helical and which contains a non-covalently attached heme group (PDB ID: 1MBN) and b) β-lactoglobulin, a 162 residue protein that has abundant β-sheet secondary structure (PDB ID: 3BLG).

8 Fig 2. Mass spectra and collision cross-sections for myoglobin and β-lactoglobulin. a, b) Mass spectra obtained when spraying from buffered (ammonium acetate, ph 7) aqueous or water/methanol solutions (green and blue, respectively). Prominent peaks are labeled, and in the case of myoglobin, they correspond to the holo form. For low charge states of β-lactoglobulin, adducts with palmitic acid can be observed (labeled 7+, 8+ and 9+ ). c, d) Cross-sections as a function of charge. Solid green circles stem from ions sprayed from buffered aqueous solution, solid blue circles from ions from

9 Amide-II 1525 cm-1 Amide-I 1655 cm-1 (helical) Amide-I 1639 cm-1 (β- sheet) Fig 3. Infrared spectra for gasphase and condensed-phase myoglobin and β-lactoglobulin. a, b)spectra for ions sprayed from water/methanol solutions. The positions and shapes of the amide-i bands around 1655 cm 1 indicate helical secondary structures. c) Spectra for samples sprayed from aqueous solutions (green lines) and solution phase FTIR spectra (gray lines; data reproduced from Ref. [18] for β-lactoglobulin and Ref. [19] for myoglobin). The amide-i band for myoglobin 8+ indicates

10 Fig S2. Circular dichroism spectra. (A) Myoglobin and (B) β-lactoglobulin either in water/methanol (blue) or buffered water (green). Dotted lines denote CD spectra of 1:1 mixtures of β-lactoglobulin and palmitic acid.

11 Conclusio n IR spectroscopy directly probes local secondary structure and can differentiate between helices and β-sheets. IR spectroscopy is thus a good complement to IMS, which on its own is blind to structural details and only probes the overall shape and size. The presented data clearly show that for β-lactoglobulin and myoglobin, the condensed-phase secondary as well as tertiary structure can be conserved when solvent is completely removed. And the combination of IR spectroscopy and IMS shown here gives a clear picture of the structural evolution of isolated proteins as their charge increases. Methods based on gas-phase mass spectrometry provide extremely high sensitivity. When mass spectrometry is combined with IMS, additionally high selectivity can be achieved through being able to select individual charge states, conformations, or aggregation states, which then can be investigated using spectroscopy. Such sensitivity and selectivity cannot be achieved using condensed-phase methods.

12 Future Perspective How these noble metal clusters are forming isomers and dimers can be studied by gas phase IR spectroscopy. May be we can get some structural insight from the IR data. In Au-Lyz work, to know the change in the secondary structure of Lyz after Au attachment, we can do gas phase IR spectroscopy.

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14 Mass/charge selected ions in helium droplets